Understanding global gene expression at the level of the whole cell requires detailed knowledge of the contributions of transcription, pre-mRNA processing, mRNA turnover, and translation. Although the sum total of these regulatory processes in each cell accounts for its unique expression profile, few methods are available to independently assess each process en masse. DNA arrays are well suited for profiling the steady-state levels of mRNA globally (i.e., the transcriptome). However, because of posttranscriptional events affecting mRNA stability and translation, the expression levels of many cellular proteins do not directly correlate with steady-state levels of mRNAs.
RNA binding proteins (RBPs) and ribonucleoprotein complexes (RNPs), such as microRNA-containing RNPs, are essential regulators of virtually all cellular activities, ranging from development, metabolism and migration to reaction to cellular stress. These proteins do so by binding to coding and non-coding RNAs at specific regions on an RNA transcript. The proteins regulate the rate of transcription, modification, splicing, nuclear export, transport, stability and translation. RNA binding proteins and RNPs recognize canonical binding motifs on a given transcript and cooperate and compete with other RBPs and RNPs in controlling its fate or metabolic rate.
A number of diseases are associated with, or caused by, deregulation or mutations in these proteins. Notable examples among autoimmune disease include systemic lupus erythematosis, primary biliary cirrhosis (PBC) and Sjogren's syndrome, and among neurologic disease include the paraneoplastic neurologic antigens Nova and Hu, and the Fragile X mental retardation FMR1 protein, the spinal muscular atrophy SMN protein, the myotonic dystrophy CELF proteins, and the spinocerebellar ataxia SCA1 protein.
Understanding the role RBPs and RNPs play in disease and normal biology, particularly in the brain, requires methods to identify the set of RNAs to which the RBPs and RNPs bind in vivo. Identifying binding motifs on the RNAs offer ways for targeted therapy. However, the targets of RBPs and RNPs involved in normal and abhorrent cellular processes and systems, including disease states such as autoimmune and genetic diseases have been difficult to identify.
Accordingly, the present invention provides methods for identifying binding sites on RNA transcripts that interact with RBPs and RNPs.